Simple boronic acids are inhibitors of serine proteases. For example, Koehler et al. Biochemistry 10: 2477 (1971) reports that 2-phenylethane boronic acid inhibits chymotrypsin at millimolar levels. The synthesis of boronic acid analogs of N-acyl-.alpha.-amino acids has yielded more effective inhibitors. Acborophe-OH, R-1-acetamido-2-phenylethane boronic acid, inhibits chymotrypsin with a K.sub.i of 4 .mu.M Matteson et al. J. Am. Chem. Soc. 103: 5241 (1981). More recently, Shenvi, U.S. Pat. No. 537,773 (1985) disclosed that boronic acid analogs of .alpha.-amino acids, containing a free amino group, were effective inhibitors of aminopeptidases. Shenvi, U.S. Pat. No. 4,499,082 (1985) discloses that peptides containing an .alpha.-aminoboronic acid with a neutral side chain were more effective inhibitors of serine proteases exceeding inhibitors disclosed earlier by as much as 3 orders of magnitude in potency. The chemistry of .alpha.-aminoboronic acids was further expanded to the synthesis of peptide analogs containing boronic acid with positive charged sidechains, boroLysine, boroArginine, boroOrnithine, and isothiouronium analogs (EPA 0 293 881, Dec. 7, 1988). This series of compounds have provided highly effective inhibitors of thrombin and other trypsin-like enzymes. The boroArginine analogs specifically designed as thrombin inhibitors are highly effective in the inhibition of blood coagulation both in vitro and in vivo.
It should be noted that additional boronic acids have been disclosed. Metternich (EP 0471651) have described peptides containing boroArginine and boroLysine which contain at least one unnatural amino acid residue. Elgendy et al. Tetrahedron Lett., 33, 4209-4212 (1992) have described peptides containing .alpha.-aminoboronic acids with aliphatic neutral sidechains which are thrombin inhibitors. Kakkar in (WO 92/07869) has claimed peptide thrombin inhibitors of the general structure, X--Aa.sub.1 --Aa.sub.2 --NH--CH(Y)--Z where Aa.sub.1 and Aa.sub.2 are unnatural amino acid residues. Z is --CN, --COR, --B(R.sup.2)(R.sup.3), --P(O)(R)(R), and Y is --[CH.sub.2 ].sub.n --Q or --CH.sub.2 --Ar--Q where Q=H, amino, amidino, imidazole, guanidino or isothioureido and n=1-5 and where R.sub.2 and R.sub.3 are the same or different and are selected from the group consisting of OH, OR.sup.6, and NR.sup.6 R.sup.7, or R.sup.2 and R.sup.3 taken together represent the residue of a diol.
Electrophilic tripeptide analogs containing the ((D-phenylalanyl)prolyl)--arginyl--sequence are well known as effective inhibitors of the trypsin-like serine protease thrombin. H-(D) Phe-Pro-ArgCH.sub.2 Cl was first reported by Kettner and Shaw (Thromb. Res. 14, 969 (1979)) to be a selective but irreversible inhibitor of human thrombin. A number of studies looking for alternatives to the electrophilic P.sub.1 argininechloromethylketones that would yield a reversible protease inhibitor have been reported. Bajuez et al. (Folia Haematol. 109, s. 16 (1982)) found the corresponding aidehyde, D-phenylalanyl-prolyl-arginal, to be a reversible thrombin inhibitor with a K.sub.i =75 nM for human thrombin. The nitrile analog, D-phenylalanyl-prolyl-NHCH((CH.sub.2).sub.3 NHC(.dbd.NH)NH.sub.2)--CN, was found to be substantially less potent with a K.sub.i =700 nM (Kaiser et al., Pharmazie 46, 128 (1991)). A retroamide inhibitor, with the D-phenylalanyl-prolyl-sequence and 2-(4-guanidinophenylalanyl)-N-acetyl-2,2-difluoroethylamine substituting for an electrophilic arginine derivative, is a good inhibitor with a K.sub.i of 70 nM for thrombin (Altenburger and Schirlin, Tetrahedron Lett. 32, 7255 (1991)). Cheng et al. claim that the substitution of racemic diphenyl 1-amino-4-methoxybutylphosphonate for an electrophilic arginine derivative gives very good inhibitors with a K.sub.i =4.8 nM (Tetrahedron Lett. 32, 7333 (1991)). Iwanowicz et al. (Bioorgan. Med. Chem. Lett. 2, 1607 (1992)) has studied the efficacy of (D-phenylalanine)prolyl- conjugated to --NHCH[(CH.sub.2).sub.4 NH.sub.2 ]CH(OH)CO.sub.2 Me) and --NHCH[(CH.sub.2).sub.4 NH.sub.2 ]C(.dbd.O)CO.sub.2 Me derivatives. The most effective inhibitor of human thrombin reported to date is the boropeptide acetyl-D-phenylalanyl-prolyl-boro arginine with a K.sub.i =0.041 nM (Kettner et al., J. Biol. Chem. 265, 18289 (1990)).
Walker et al. (Biochem. J. 230, 645 (1985)) published a comparative study of irreversible thrombin inhibitors based on the D-phenylalanyl-prolyl-argininyl sequence confirming the earlier report by Kettner and Shaw (1979). H-(D) Phe-Pro-ArgCH.sub.2 Cl was found to be the most effective inhibitor (K.sub.i =25 nM) while replacing the D-phenylalanine with 4-amino-D-phenylalanine or .omega.-benzoyl-D-lysine gave less active analogs. Compounds in development include -(prolyl)arginal derivatives with a variety of unusual P.sub.3 amino acids including D-N-methylphenylglycine, Boc-D-fluorophenylglycine as well as constrained cyclized derivatives of D-phenylglycine and D-phenylalanine (Shuman et. al., J. Med. Chem. 36, 314 (1993)). Balasubramanian et al. (J. Med. Chem. 36, 300 (1993)) has reported an extensive study of replacements for the P.sub.3 D-phenylalanine of D-phenylalanyl-prolyt-arginal and found the dihydrocinnamoyl group to be effective, although somewhat less potent.
Patent disclosures in this area have centered around suitably protected peptides composed of natural and unnatural amino acids. In U.S. Pat. No. 5,187,157 DuPont Merck has disclosed peptides comprised of C-terminal boronic acid derivatives of lysine, ornithine and arginine as reversible inhibitors of trypsin-like serine proteases. In European Patent Application EP 471 651 A2 Sandoz disclosed borolysine and boroarginine peptide analogs containing at least one unnatural hydrophobic .alpha.-amino acid substituted with groups such as the trimethylsilyl- or naphthyl-. In U.S. Pat. No. 5,106,948 was disclosed a series of boropeptides that are effective as cytotoxic agents. In PCT Application WO 92/07869, Thrombosis Research Institute has disclosed tripeptide analogs containing a P.sub.2 proline and an unnatural disubstituted amino acid at P.sub.3. A variety of electrophilic and non-electrophilic .alpha.-amino acid analogs were claimed as suitable P.sub.1 substituents. Tripeptide antithrombotic agents limited to .alpha.-alkyl and .alpha.-aryl or heteroaryl substituted glycines at P.sub.3 conjugated to -(prolyl)arginal were disclosed by Lilly (European Patent Application EP 479 489 A2). Marion Merrell Dow disclosed a series of activated electrophilic ketone analogs of peptidase substrates useful for inhibiting serine-, carboxylic acid- and metallo- proteolytic enzymes; compounds are peptides composed of suitably protected .alpha.-amino acids conjugated to an electrophilic ketone derivative of an .alpha.-amino acid (European Patent Applications EP 417 721 A2, EP 364 344 A2, EP 363 284 A2, EP 195 212 A2). Astra has disclosed a series of .alpha.-((trifluoroethyl)oxymethyl)-arginine tripeptides (European Patent Application EP 0 530 167 A). Georgia Tech Research Corporation disclosed peptidyl ketoamides, -ketoacids and -ketoesters as inhibitors of serine and cysteine proteases (WO 92/12140). Boehringer Ingelheim disclosed a series of trifluoromethyl- and .alpha.,.alpha.-difluoromethyl-.beta.-ketoesterpeptide derivatives as elastase inhibitors (EP 0 369 391 A2).
A number of naturally occurring thrombin inhibitors have been reported. These include nazumamide A from Theonella sp. (see Fusetani, et. al., Tetrahedron Lett. 1991, 32, 7073-4), cyclotheonamide A from Theonelia sp. (see Fusetani, et. al., J. Am. Chem. Soc. 1990, 112, 7053-4 (1990)), amblyommin from Amblyomma hebraeum (see Bonin, et. al., EP 345614), hirudin from Hirudo medicinalis, recombinant versions of hirudin and hirudin fragments (see Rigbl and Jackson, EP 352903, Koerwer, WO 9109946, Meyer, et. al., WO 9108233, Dawson, et. al., WO 9109125, Maraganore, et. al., WO 9102750 and Maraganore, EP 333356).
Synthetic thrombin inhibitors have also been disclosed. Arylsulfonylarginine amides such as (2R,2R)-4-methyl-1-[N.sup.2 -{(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl) sulfonyl}-L-arginyl]-2-piperidinecarboxylate have been shown to be effective inhibitors of thrombin (see Okamoto, et. al. Thromb Res., 8, 77-82 (1976), Ohshiro, et. al., Blood Vessel, 14, 216-8 (1983)), as have compounds containing constrained arginine mimics such as (2-naphthylsulfonylglycyl)-4-amidino-phenylalanyl piperidide (see Stuerzebecher, et. al., Thromb. Res., 29, 635-42 (1983)), 1-[2-[5-(dimethylamino)naphth-1-ylsulfonamido]-3-(2-iminohexahydropyrimidi n-5-yl)propanoyl]-4-methylpiperidine dihydrochloride (see Ishikawa, JP 88227572 and Ishikawa and Inamura, JP 88227573), N-(trans-4-amino-methylcyclohexylcarbonyl)-4-O-(2-picolyl)-L-tyrosine 4-acetanilide dihydrochloride (see Okamoto, et. al., EP 217286) and 4-[(aminoiminomethyl)amino]benzoic acid esters (see Fuji, et. al., DE 3005580, Matsuoka, et. al., Jpn. J. Pharmacol., 51, 455-63 (1989), and Takeshita, et. al., EP 435235).
Inhibitor design has benefitted from the knowledge of the mechanism of action and of the peptide sequences which are thought to bind in the catalytic site of thrombin, e.g., -Gly-Vat-Arg-Gly- of fibrinogen (see Blomback, et. al., J. Biol. Chem., 247, 1496-512 (1972)), Ile-Pro-Arg-Ser- of prothrombin (see Magnussen, et. al., in: Reich, et. al., "Proteases and Biological Control," pp. 123-149 (1975)) and -Val-Pro-Arg-Gly- of factor XIII (see Takagi and Doolittle, Biochemistry, 13, 750-6 (1974), and Nakamura, et. al., Biochem. Biophys. Res. Commun., 58, 250-256 (1974)). This class of mechanism-based inhibitors are exemplified by the tripeptide aldehyde D-Phe-Pro-N-Me-Arg-H (see Bajuez, et. al., J. Med. Chem., 33, 1729-35 (1990)), and the trifluoromethyl ketone D-Phe-Pro-ArgCF.sub.3 (see Kolb, et. al., U.S. Pat. No. 697987).
Surprising for their lack of a basic residue at P.sub.1 are tripeptide thrombin inhibitors comprised of 1-aminoboronic and 1-aminophosphonic acid analogs of 3-methoxy-propylglycine (see Claeson, et. al., U.S. 07-245,428) and pentylglycine (see Cheng, et. al., "Symposium on Thrombosis and Hemostasis,"1991, Amsterdam, Abstract 2150).
In addition to thrombin inhibition, boropeptides have been disclosed with utility as a treatment for tumors, viral infections and arthritis (US 4963655A and EP 354522A), hypertension (EP 315574A) and as factor VII/VIIa inhibitors (WO 8909612A). Kleemann, et. al. (AU A-24693/88) disclose renin-inhibiting 1-amino boronic acid derivatives of formula (3) EQU A.sup.1 --A.sup.2 --HN--CHR.sup.2 --BXR.sup.3 (YR.sup.4) (3)
in which A.sup.1 denotes a radical of formulae (4-8). EQU R.sup.1 NR.sup.6 --CHR.sup.5 --C.dbd.O-- (4) EQU R.sup.1 CHR.sup.12 --CHR.sup.5 --C.dbd.O-- (5) EQU R.sup.1 NR.sup.6 --CHR.sup.5 --CHR.sup.7 --CHR.sup.8 --CHR.sup.9 --C.dbd.O--(6) EQU R.sup.1 CHR.sup.12 --CHR.sup.5 --CHR.sup.7 --CHR.sup.8 --CHR.sup.9 --C.dbd.O-- (7) EQU R.sup.10 --(CH.sub.2).sub.n --CH(CH.sub.2).sub.m R.sup.11 --C.dbd.O--(8)
A recently issued Patent (U.S. Pat. No. 5,332,822, issued Jul. 26, 1994, R. N. Misra) claims compounds of the general formula (9) which are useful as thrombin inhibitors. Substituent definitions are as follows: R.sup.1 and R.sup.2 are independently H, lower alkyl, cycloalkyl, aryl, heteroaryl, or heteroarylalkyl; or together R.sup.1 and R.sup.2 are a heterocycle containing N, S, and/or O optionally substituted with lower alkyl, carboxy, amido, carboalkoxy, aryl, cycloalkyl, hydroxy, amino, alkylamino, or dialkylamino. R.sup.3 is heteroaryl. R.sup.4 is alkyl, cycloalkyl, aryl, tetrahydronaphthyl, heteroaryl, quinolinyl, or tetrahydroquinolinyl. No biological data is disclosed. ##STR2##
Despite the foregoing, more efficacious and specific inhibitors of coagulation proteases are needed as potentially valuable therapeutic agents for the treatment of thrombosis. None of the cited references describe or suggest the new thrombin-inhibiting boronic acid derivatives of the present invention.
The present invention concerns dipeptides which contain an electrophilic derivative of an .alpha.-amino acid at P.sub.1 (where P.sub.1 is the carboxyl terminus of the dipeptide). The P.sub.1 substituent contains an optionally functionalized imidazole group, and the P1 is conjugated to an N,N-disubstituted or N-monosubstituted .alpha.-amino acid at P.sub.2 (where P.sub.2 is the end-terminus of the dipeptide). The electrophilic functional groups used to derivatize the P.sub.1 amino acid analog are boronic acids and their esters. The N,N-disubstituted .alpha.-amino acids are derivatives of an amino acid other than proline where the .alpha.-amino group is alkylated and acylated or diacylated to give alicyclic or cyclic substituents.